Project 3 Abstract Metabolic regulation of human erythropoiesis The self renewal capacity of hematopoietic stem cells (HSCs) is controlled by the cells' metabolic state but the possibility that nutrient entry and metabolism contribute to the differential commitment of an HSC to a lymphoid, myeloid or erythroid lineage fate was not considered until very recently. The overall goal of this project is to develop a mechanistic understanding of the role of cell metabolism in physiological and disordered erythropoiesis. Our studies address the hypothesis that nutrient transport and utilization regulate both normal and pathological human erythroid differentiation. Our previous data show that the glucose transporter Glut1 is only upregulated during the final mitoses of human erythroid differentiation (Montel- Hagen et al. Cell 2008) whereas the glutamine transporter ASCT2 is expressed on all HSCs. We determined that down regulation of ASCT2 or blocking glutamine metabolism abrogates erythroid differentiation and skews erythropoietin-treated HSCs towards a myeloid fate. In contrast, diverting glucose into the pentose phosphate pathway, away from glycolysis, accelerates erythropoiesis (Oburoglu et al. Cell Stem Cell 2014). In Aim 1, we will use our unique collection of retroviral envelope receptor binding domains (RBDs), that function as specific ligands of solute carrier (SLC) nutrient transporters, to characterize stage- specific expression and function of transporters and determine the array of transporters regulating erythropoiesis in normal conditions as well as in erythroid progenitors with altered nuclear lamins (with Project 4), in a TET2-deficient model of myelodysplastic syndrome, and in RPL5- and RPL11-deficient models of Diamond Blackfan anemia (with Project 1). In Aim 2, we will assess metabolic fluxes from stable glucose, glutamine, and fatty acid isotope tracers, elucidating the metabolic networks and metabolites that regulate normal and perturbed erythropoiesis. These studies will critically address our hypothesis that fuel resource utilization governs early and terminal erythroid differentiation, at a level beyond simply providing the ATP, amino acids and lipids that are required for cell division. We propose that metabolic changes contribute to stage-specific epigenetic, transcriptional and translational erythroid regulatory programs which will be evaluated with Project 2. We anticipate that integration of these data within the Program Project will identify the nutrient fluxes and utilization that control stage-specific erythroid transitions, pioneer nutrient transporter biomarker discovery in erythroid disorders, and promote the manipulation of nutrient transporters and metabolic networks that orient physiological and pathological erythroid cell differentiation and survival.